RESEARCH ARTICLE
Drinking Citrus Fruit Juice Inhibits Vascular Remodeling in Cuff-Induced Vascular Injury Mouse Model Arika Ohnishi1‡, Rie Asayama1‡, Masaki Mogi1*, Hirotomo Nakaoka1, Harumi Kan-no1, Kana Tsukuda1, Toshiyuki Chisaka1,2, Xiao-Li Wang1, Hui-Yu Bai1, Bao-Shuai Shan1, Masayoshi Kukida1,3, Jun Iwanami1, Masatsugu Horiuchi1 1 Department of Molecular Cardiovascular Biology and Pharmacology, Ehime University, Graduate School of Medicine, Tohon, Ehime, Japan, 2 Department of Pediatrics, Ehime University, Graduate School of Medicine, Tohon, Ehime, Japan, 3 Department of Cardiology, Pulmonology, Hypertension and Nephrology, Ehime University, Graduate School of Medicine, Tohon, Ehime, Japan ‡ These authors contributed equally to this work. * [email protected]
OPEN ACCESS Citation: Ohnishi A, Asayama R, Mogi M, Nakaoka H, Kan-no H, Tsukuda K, et al. (2015) Drinking Citrus Fruit Juice Inhibits Vascular Remodeling in CuffInduced Vascular Injury Mouse Model. PLoS ONE 10 (2): e0117616. doi:10.1371/journal.pone.0117616 Received: September 4, 2014 Accepted: December 29, 2014 Published: February 18, 2015 Copyright: © 2015 Ohnishi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This study was supported by JSPS KAKENHI Grant Numbers 25293310 (MH) and 25462220 (MM), and research grants from pharmaceutical companies: Astellas Pharma Inc., Bayer Yakuhin, Ltd., Daiichi-Sankyo Pharmaceutical Co. Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Shionogi & Co., Ltd., and Takeda Pharmaceutical Co. Ltd. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Abstract Citrus fruits are thought to have inhibitory effects on oxidative stress, thereby attenuating the onset and progression of cancer and cardiovascular disease; however, there are few reports assessing their effect on vascular remodeling. Here, we investigated the effect of drinking the juice of two different citrus fruits on vascular neointima formation using a cuffinduced vascular injury mouse model. Male C57BL6 mice were divided into five groups as follows: 1) Control (water) (C), 2) 10% Citrus unshiu (CU) juice (CU10), 3) 40% CU juice (CU40), 4) 10% Citrus iyo (CI) juice (CI10), and 5) 40% CI juice (CI40). After drinking them for 2 weeks from 8 weeks of age, cuff injury was induced by polyethylene cuff placement around the femoral artery. Neointima formation was significantly attenuated in CU40, CI10 and CI40 compared with C; however, no remarkable preventive effect was observed in CU10. The increases in levels of various inflammatory markers including cytokines such as monocyte chemotactic protein-1, interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α in response to vascular injury did not differ significantly between C, CU10 and CI10. The increases in cell proliferation and superoxide anion production were markedly attenuated in CI10, but not in CU10 compared with C. The increase in phosphorylated ERK expression was markedly attenuated both in CU10 and CI10 without significant difference between CU10 and CI10. Accumulation of immune cells did not differ between CU10 and CI10. These results indicate that drinking citrus fruit juice attenuates vascular remodeling partly via a reduction of oxidative stress. Interestingly, the preventive efficacy on neointima formation was stronger in CI than in CU at least in part due to more prominent inhibitory effects on oxidative stress by CI.
Competing Interests: This study was supported by JSPS KAKENHI Grant Numbers 25293310 (MH) and 25462220 (MM), and research grants from
PLOS ONE | DOI:10.1371/journal.pone.0117616 February 18, 2015
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Drinking Citrus Fruit Juice Inhibits Vascular Remodeling
pharmaceutical companies: Astellas Pharma Inc., Bayer Yakuhin, Ltd., Daiichi-Sankyo Pharmaceutical Co. Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Shionogi & Co., Ltd., and Takeda Pharmaceutical Co. Ltd. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.
Introduction Dietary habits are closely related to most lifestyle-related diseases such as cardiovascular disease (CVD). Therefore, dietary modification is one of the cornerstones for prevention of the onset of CVD. High consumption of fruit is reported to be beneficial for reduction of cardiovascular mortality [1], prevention of ischemic heart disease [2] and stroke [3], and blood pressure lowering [4,5,6,7]. Among them, eating citrus fruits and/or drinking their juice have preventive effects on the pathophysiology of ischemic stroke [8] and CVD etc. [9] Citrus fruits are one of the most popular and highly consumed type of fruit in the United States and Japan. Citrus unshiu (also known as Mandarin orange or Mikan) is the most popular citrus fruit and ingredient of fruit juice in Japan. Moreover, Citrus iyo (also known as iyokan) is the second most widely produced citrus fruit in Japan. Recently, the effects of consumption of Citrus unshiu on cognitive function [10], nitric oxide production [11] and allergic rhinitis [12] have been reported, suggesting that Citrus unshiu intake could have beneficial effects on the pathophysiology of cardiovascular disease. Buscemi et al. recently demonstrated that drinking red orange juice for seven days ameliorated endothelial dysfunction and reduced inflammation in non-diabetic subjects with increased cardiovascular risk [13]. Moreover, Codoñer-Franch et al. reported that drinking mandarin juice showed an anti-oxidant effect in children [14,15] and rats [16]. However, red orange juice is not popular in Japan, and the effect of drinking citrus juice on vascular function has never been investigated using animal models. Therefore, we investigated the effect of drinking citrus juice on vascular remodeling using a cuff-induced vascular injury model in mice, and we also examined the possible difference in vascular protective effects between Citrus unshiu and Citrus iyo.
Methods All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and reviewed and approved by the Animal Studies Committee of Ehime University.
Citrus Fruit Juice Preparation The juice of Citrus unshiu (CU) and Citrus iyo (CI) was prepared and provided by Ehime Beverage Inc. (Ehime, Japan). Citrus juice was prepared as follows. Juice was extracted from washed fresh CU or CI by in-line juice extractor (John Bean Technologies Corp., Chicago, IL). Using a hydro-cyclone between the procedures of extractors and finishers, the defects such as embryonic seeds were removed. After treatment with finishers, juice was centrifuged at 7,500 x g to remove the part of insoluble solids. The juice was filled into bottles followed pasteurization at 96°C for 30 seconds and cooling. The bottles were kept in the cold storage at -18°C before use.
Animals and Treatment Male C57BL6 mice (CLEA, Tokyo, Japan) were divided into five groups as follows: 1) Control (water) (C), 2) 10% CU (CU10), 3) 40% CU (CU40), 4) 10% CI (CI10), and 5) 40% CI (CI40). The animals were housed in a room in which lighting was controlled (12 hours on and 12 hours off), and room temperature was kept at 25°C. They were given a standard diet (MF, Oriental Yeast Co., Ltd., Tokyo, Japan). After drinking juice ad libitum for 2 weeks from 8 weeks of age, cuff injury was induced by polyethylene cuff placement around the femoral artery. After 2 weeks of drinking citrus fruit juice, systolic blood pressure was measured by the tail-cuff method (MK-2000ST, Muromachi Kikai, Co. Ltd., Tokyo, Japan) as described previously [17].
PLOS ONE | DOI:10.1371/journal.pone.0117616 February 18, 2015
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Drinking Citrus Fruit Juice Inhibits Vascular Remodeling
Cuff-induced Vascular Injury Model Adult male C57BL/6J mice (10 weeks of age) were used in this study. Inflammatory cuff injury was induced by polyethylene cuff placement around the femoral artery under anesthesia with intraperitoneal injection of 60 mg/kg pentobarbital sodium in saline, and morphometric analysis to measure the neointimal area was performed as described previously [18,19].
Immunohistochemical Staining Formalin-fixed, paraffin-embedded sections were prepared using femoral artery 7 days after cuff placement. Endogenous peroxidase was blocked by incubation in 3% H2O2 for 15 min, and nonspecific protein binding was blocked by incubation for 10 min in Blocking Reagent (Nichirei Bioscience Inc., Tokyo, Japan). The sections were incubated overnight at 4°C with the primary antibody, anti-proliferating cell nuclear antigen (PCNA) antibody (Novocastra Laboratories, Ltd., Newcastle upon Tyne, UK), and phosphorylated extracellular signal-regulated kinase (ERK) and total ERK antibodies (Cell Signaling Technology, Pickering, ON) and Alexa Fluor 488-conjugated F4/80 (BioLegend Inc., San Diego, CA) and R-PE-conjugated LY-6G/6C (Hycult Biotech Inc., Plymouth Meeting, PA). Antibody binding was visualized by followings: 1) a Zeiss Axioskop2 microscope (Carl Zeiss, Oberkochen, Germany) equipped with a computer-based imaging system for 3, 3’-diaminobenzidine (DAB) staining using a detection kit, Histofine (Nichirei Bioscience Inc.) for PCNA and ERK staining, and 2) a fluorescence microscope (Keyence BZ-9000, Osaka, Japan) equipped with a computer-based imaging system for immunofluorescent staining.
Real Time RT-PCR Total RNA was extracted from a pool of four different femoral arteries. Real-time quantitative reverse-transcription polymerase chain reaction (PCR) was performed with a SYBR Premix Ex Taq (Takara Bio Inc., Shiga, Japan). PCR primers were as follows: MCP-1, 5’-TTAACGCCCC ACTCACCTGCTG-3’ (forward) and 5’-GCTTCTTTGGGACACCTGCTGC-3’ (reverse); tumor necrosis factor-α (TNF-α), 5’-CGAGTGACAAGCCTGTAGCC-3’ (forward) and 5’-GGTGAGG AGCACGATGTCG-3’ (reverse); IL-6, 5’-CCACTTCACAAGTCGGAGGCTTA-3’ (forward) and 5’-GCAAGTGCATCATCGTTGTTCATAC-3’ (reverse); interleukin-1β (IL-1β), 5’-TCC AGGATGAGGACATGAGCAC-3’ (forward) and 5’-GAACGTCACACACCAGCAGGTTA-3’ (reverse); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5’-ATGTAGGCCATGAGG TCCAC-3’ (forward) and 5’-TGCGACTTCAACAGCAACTC-3’ (reverse).
Dihydroethidium Staining Superoxide generation in cryostat frozen sections was evaluated using fluorogenic dihydroethidium (5 μmol/L), as described previously [20]. Intensity of fluorescence was analyzed and quantified using computer imaging software (Densitograph, ATTO Corp., Tokyo, Japan).
Immunoblot Analysis Femoral arteries were obtained 7 days after cuff placement and the adventitia was carefully removed. The extracted proteins from the femoral arteries were subjected to SDS-PAGE and immunoblotted with rabbit anti-phosphorylated ERK and total ERK antibodies (Cell Signaling Technology). The bands of proteins were visualized using an enhanced chemiluminescence system (GE Healthcare, Buckinghamshire, England).
PLOS ONE | DOI:10.1371/journal.pone.0117616 February 18, 2015
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Drinking Citrus Fruit Juice Inhibits Vascular Remodeling
Statistical Analysis All values are expressed as mean ± S.D. in the text and figures. Data were evaluated by ANOVA. If a statistically significant effect was found, post hoc analysis was performed to detect the difference between the groups. Values of P < 0.05 were considered to be statistically significant.
Results Inhibitory Effect of Citrus Fruits on Neointima Formation We examined the effects of drinking citrus fruit juice on neointima formation 14 days after polyethylene cuff placement around the femoral artery, and observed that drinking citrus fruit juice attenuated neointima formation (Fig. 1A and 1B) without a change in body weight, blood glucose or blood pressure (S1 Fig). Neointima formation was significantly attenuated in CU40, CI10 and CI40 compared with C; however, no remarkable preventive effect was observed in CU10. During the experimental period, no significant change in fluid intake was observed in each group (S2 Fig). To assess the more detailed mechanism of the differences between CU and CI in terms of the inhibitory effect on neointimal formation and cell proliferation, we focused on three groups: control, CU10 and CI10.
Inhibitory Effect of Citrus Fruits on Cell Proliferation We observed that drinking citrus fruit juice decreased the neointima area, with a decrease in PCNA labeling index in both the intima and media (Fig. 2A and 2B). The increase in cell proliferation after cuff-placement was markedly attenuated in CI10, but not in CU10 compared with C.
Inflammatory Cytokine Levels Were Not Attenuated by Citrus Fruits We assessed the effect of drinking citrus fruit juice on mRNA levels of inflammatory cytokines in the femoral artery 7 days after cuff placement (Fig. 3). The increases in various inflammatory cytokines such as monocyte chemotactic protein-1, interleukin-6 (IL-6), IL-1β and tumor necrosis factor-α in response to vascular injury did not differ significantly among C, CU10 and CI10.
Inhibitory Effect of Citrus Fruits on Superoxide Anion Production Next, we examined the effect of drinking citrus fruit juice on superoxide anion production in the femoral artery 7 days after cuff placement (Fig. 4A and 4B). The increase in superoxide anion production determined by dihydroethidium staining was markedly attenuated in CI10, but not in CU10 compared with C.
Inhibitory Effect of Citrus Fruits on ERK Activation We investigated ERK activation in the injured artery 7 days after cuff placement (Fig. 5). The increase in expression of phosphorylated ERK determined by immunohistochemical staining and immunoblot using anti-phosphorylated ERK antibody. The increase in phosphorylated ERK positive cells in neointima determined by immunohistochemical staining was markedly attenuated both in CU10 and CI10 compared with C (Fig. 5A and 5B). Consistent with these immunohistochemical results, ERK activity determined with Western blotting was markedly attenuated in CI10 and CU10 compared with C (Fig. 5C). However, significant difference in the inhibitory effects between CU10 and CI10 on ERK activation was not observed.
PLOS ONE | DOI:10.1371/journal.pone.0117616 February 18, 2015
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Drinking Citrus Fruit Juice Inhibits Vascular Remodeling
Fig 1. Effect of drinking citrus fruit juice on neointima formation. Male C57BL6 mice were divided into five groups as follows: 1) Control (water) (C), 2) 10% Citrus unshiu (CU) juice (CU10), 3) 40% CU juice (CU40), 4) 10% Citrus iyo (CI) juice (CI10), and 5) 40% CI juice (CI40). After drinking them for 2 weeks from 8 weeks of age, cuff injury was induced by polyethylene cuff placement around the femoral artery. Samples were prepared from cuffed-femoral arteries of C57BL/6J mice as described in Methods. A, Representative photos of neointimal area in cross-sections of femoral artery with elastic van Gieson staining 14 days after cuff placement at 100x magnification. B, Higher magnified photos at 400x magnification described as squares in Figure A. Scale bars show 50 μm in each photo. C, Quantitative analysis of neointimal area in injured femoral artery. Values are mean ± SEM (n = 6 for Cuff (-), n = 8 for other groups). *p
Drinking Citrus Fruit Juice Inhibits Vascular Remodeling in Cuff-Induced Vascular Injury Mouse Model Arika Ohnishi1‡, Rie Asayama1‡, Masaki Mogi1*, Hirotomo Nakaoka1, Harumi Kan-no1, Kana Tsukuda1, Toshiyuki Chisaka1,2, Xiao-Li Wang1, Hui-Yu Bai1, Bao-Shuai Shan1, Masayoshi Kukida1,3, Jun Iwanami1, Masatsugu Horiuchi1 1 Department of Molecular Cardiovascular Biology and Pharmacology, Ehime University, Graduate School of Medicine, Tohon, Ehime, Japan, 2 Department of Pediatrics, Ehime University, Graduate School of Medicine, Tohon, Ehime, Japan, 3 Department of Cardiology, Pulmonology, Hypertension and Nephrology, Ehime University, Graduate School of Medicine, Tohon, Ehime, Japan ‡ These authors contributed equally to this work. * [email protected]
OPEN ACCESS Citation: Ohnishi A, Asayama R, Mogi M, Nakaoka H, Kan-no H, Tsukuda K, et al. (2015) Drinking Citrus Fruit Juice Inhibits Vascular Remodeling in CuffInduced Vascular Injury Mouse Model. PLoS ONE 10 (2): e0117616. doi:10.1371/journal.pone.0117616 Received: September 4, 2014 Accepted: December 29, 2014 Published: February 18, 2015 Copyright: © 2015 Ohnishi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This study was supported by JSPS KAKENHI Grant Numbers 25293310 (MH) and 25462220 (MM), and research grants from pharmaceutical companies: Astellas Pharma Inc., Bayer Yakuhin, Ltd., Daiichi-Sankyo Pharmaceutical Co. Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Shionogi & Co., Ltd., and Takeda Pharmaceutical Co. Ltd. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Abstract Citrus fruits are thought to have inhibitory effects on oxidative stress, thereby attenuating the onset and progression of cancer and cardiovascular disease; however, there are few reports assessing their effect on vascular remodeling. Here, we investigated the effect of drinking the juice of two different citrus fruits on vascular neointima formation using a cuffinduced vascular injury mouse model. Male C57BL6 mice were divided into five groups as follows: 1) Control (water) (C), 2) 10% Citrus unshiu (CU) juice (CU10), 3) 40% CU juice (CU40), 4) 10% Citrus iyo (CI) juice (CI10), and 5) 40% CI juice (CI40). After drinking them for 2 weeks from 8 weeks of age, cuff injury was induced by polyethylene cuff placement around the femoral artery. Neointima formation was significantly attenuated in CU40, CI10 and CI40 compared with C; however, no remarkable preventive effect was observed in CU10. The increases in levels of various inflammatory markers including cytokines such as monocyte chemotactic protein-1, interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α in response to vascular injury did not differ significantly between C, CU10 and CI10. The increases in cell proliferation and superoxide anion production were markedly attenuated in CI10, but not in CU10 compared with C. The increase in phosphorylated ERK expression was markedly attenuated both in CU10 and CI10 without significant difference between CU10 and CI10. Accumulation of immune cells did not differ between CU10 and CI10. These results indicate that drinking citrus fruit juice attenuates vascular remodeling partly via a reduction of oxidative stress. Interestingly, the preventive efficacy on neointima formation was stronger in CI than in CU at least in part due to more prominent inhibitory effects on oxidative stress by CI.
Competing Interests: This study was supported by JSPS KAKENHI Grant Numbers 25293310 (MH) and 25462220 (MM), and research grants from
PLOS ONE | DOI:10.1371/journal.pone.0117616 February 18, 2015
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Drinking Citrus Fruit Juice Inhibits Vascular Remodeling
pharmaceutical companies: Astellas Pharma Inc., Bayer Yakuhin, Ltd., Daiichi-Sankyo Pharmaceutical Co. Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novartis Pharma K.K., Shionogi & Co., Ltd., and Takeda Pharmaceutical Co. Ltd. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.
Introduction Dietary habits are closely related to most lifestyle-related diseases such as cardiovascular disease (CVD). Therefore, dietary modification is one of the cornerstones for prevention of the onset of CVD. High consumption of fruit is reported to be beneficial for reduction of cardiovascular mortality [1], prevention of ischemic heart disease [2] and stroke [3], and blood pressure lowering [4,5,6,7]. Among them, eating citrus fruits and/or drinking their juice have preventive effects on the pathophysiology of ischemic stroke [8] and CVD etc. [9] Citrus fruits are one of the most popular and highly consumed type of fruit in the United States and Japan. Citrus unshiu (also known as Mandarin orange or Mikan) is the most popular citrus fruit and ingredient of fruit juice in Japan. Moreover, Citrus iyo (also known as iyokan) is the second most widely produced citrus fruit in Japan. Recently, the effects of consumption of Citrus unshiu on cognitive function [10], nitric oxide production [11] and allergic rhinitis [12] have been reported, suggesting that Citrus unshiu intake could have beneficial effects on the pathophysiology of cardiovascular disease. Buscemi et al. recently demonstrated that drinking red orange juice for seven days ameliorated endothelial dysfunction and reduced inflammation in non-diabetic subjects with increased cardiovascular risk [13]. Moreover, Codoñer-Franch et al. reported that drinking mandarin juice showed an anti-oxidant effect in children [14,15] and rats [16]. However, red orange juice is not popular in Japan, and the effect of drinking citrus juice on vascular function has never been investigated using animal models. Therefore, we investigated the effect of drinking citrus juice on vascular remodeling using a cuff-induced vascular injury model in mice, and we also examined the possible difference in vascular protective effects between Citrus unshiu and Citrus iyo.
Methods All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and reviewed and approved by the Animal Studies Committee of Ehime University.
Citrus Fruit Juice Preparation The juice of Citrus unshiu (CU) and Citrus iyo (CI) was prepared and provided by Ehime Beverage Inc. (Ehime, Japan). Citrus juice was prepared as follows. Juice was extracted from washed fresh CU or CI by in-line juice extractor (John Bean Technologies Corp., Chicago, IL). Using a hydro-cyclone between the procedures of extractors and finishers, the defects such as embryonic seeds were removed. After treatment with finishers, juice was centrifuged at 7,500 x g to remove the part of insoluble solids. The juice was filled into bottles followed pasteurization at 96°C for 30 seconds and cooling. The bottles were kept in the cold storage at -18°C before use.
Animals and Treatment Male C57BL6 mice (CLEA, Tokyo, Japan) were divided into five groups as follows: 1) Control (water) (C), 2) 10% CU (CU10), 3) 40% CU (CU40), 4) 10% CI (CI10), and 5) 40% CI (CI40). The animals were housed in a room in which lighting was controlled (12 hours on and 12 hours off), and room temperature was kept at 25°C. They were given a standard diet (MF, Oriental Yeast Co., Ltd., Tokyo, Japan). After drinking juice ad libitum for 2 weeks from 8 weeks of age, cuff injury was induced by polyethylene cuff placement around the femoral artery. After 2 weeks of drinking citrus fruit juice, systolic blood pressure was measured by the tail-cuff method (MK-2000ST, Muromachi Kikai, Co. Ltd., Tokyo, Japan) as described previously [17].
PLOS ONE | DOI:10.1371/journal.pone.0117616 February 18, 2015
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Drinking Citrus Fruit Juice Inhibits Vascular Remodeling
Cuff-induced Vascular Injury Model Adult male C57BL/6J mice (10 weeks of age) were used in this study. Inflammatory cuff injury was induced by polyethylene cuff placement around the femoral artery under anesthesia with intraperitoneal injection of 60 mg/kg pentobarbital sodium in saline, and morphometric analysis to measure the neointimal area was performed as described previously [18,19].
Immunohistochemical Staining Formalin-fixed, paraffin-embedded sections were prepared using femoral artery 7 days after cuff placement. Endogenous peroxidase was blocked by incubation in 3% H2O2 for 15 min, and nonspecific protein binding was blocked by incubation for 10 min in Blocking Reagent (Nichirei Bioscience Inc., Tokyo, Japan). The sections were incubated overnight at 4°C with the primary antibody, anti-proliferating cell nuclear antigen (PCNA) antibody (Novocastra Laboratories, Ltd., Newcastle upon Tyne, UK), and phosphorylated extracellular signal-regulated kinase (ERK) and total ERK antibodies (Cell Signaling Technology, Pickering, ON) and Alexa Fluor 488-conjugated F4/80 (BioLegend Inc., San Diego, CA) and R-PE-conjugated LY-6G/6C (Hycult Biotech Inc., Plymouth Meeting, PA). Antibody binding was visualized by followings: 1) a Zeiss Axioskop2 microscope (Carl Zeiss, Oberkochen, Germany) equipped with a computer-based imaging system for 3, 3’-diaminobenzidine (DAB) staining using a detection kit, Histofine (Nichirei Bioscience Inc.) for PCNA and ERK staining, and 2) a fluorescence microscope (Keyence BZ-9000, Osaka, Japan) equipped with a computer-based imaging system for immunofluorescent staining.
Real Time RT-PCR Total RNA was extracted from a pool of four different femoral arteries. Real-time quantitative reverse-transcription polymerase chain reaction (PCR) was performed with a SYBR Premix Ex Taq (Takara Bio Inc., Shiga, Japan). PCR primers were as follows: MCP-1, 5’-TTAACGCCCC ACTCACCTGCTG-3’ (forward) and 5’-GCTTCTTTGGGACACCTGCTGC-3’ (reverse); tumor necrosis factor-α (TNF-α), 5’-CGAGTGACAAGCCTGTAGCC-3’ (forward) and 5’-GGTGAGG AGCACGATGTCG-3’ (reverse); IL-6, 5’-CCACTTCACAAGTCGGAGGCTTA-3’ (forward) and 5’-GCAAGTGCATCATCGTTGTTCATAC-3’ (reverse); interleukin-1β (IL-1β), 5’-TCC AGGATGAGGACATGAGCAC-3’ (forward) and 5’-GAACGTCACACACCAGCAGGTTA-3’ (reverse); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5’-ATGTAGGCCATGAGG TCCAC-3’ (forward) and 5’-TGCGACTTCAACAGCAACTC-3’ (reverse).
Dihydroethidium Staining Superoxide generation in cryostat frozen sections was evaluated using fluorogenic dihydroethidium (5 μmol/L), as described previously [20]. Intensity of fluorescence was analyzed and quantified using computer imaging software (Densitograph, ATTO Corp., Tokyo, Japan).
Immunoblot Analysis Femoral arteries were obtained 7 days after cuff placement and the adventitia was carefully removed. The extracted proteins from the femoral arteries were subjected to SDS-PAGE and immunoblotted with rabbit anti-phosphorylated ERK and total ERK antibodies (Cell Signaling Technology). The bands of proteins were visualized using an enhanced chemiluminescence system (GE Healthcare, Buckinghamshire, England).
PLOS ONE | DOI:10.1371/journal.pone.0117616 February 18, 2015
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Drinking Citrus Fruit Juice Inhibits Vascular Remodeling
Statistical Analysis All values are expressed as mean ± S.D. in the text and figures. Data were evaluated by ANOVA. If a statistically significant effect was found, post hoc analysis was performed to detect the difference between the groups. Values of P < 0.05 were considered to be statistically significant.
Results Inhibitory Effect of Citrus Fruits on Neointima Formation We examined the effects of drinking citrus fruit juice on neointima formation 14 days after polyethylene cuff placement around the femoral artery, and observed that drinking citrus fruit juice attenuated neointima formation (Fig. 1A and 1B) without a change in body weight, blood glucose or blood pressure (S1 Fig). Neointima formation was significantly attenuated in CU40, CI10 and CI40 compared with C; however, no remarkable preventive effect was observed in CU10. During the experimental period, no significant change in fluid intake was observed in each group (S2 Fig). To assess the more detailed mechanism of the differences between CU and CI in terms of the inhibitory effect on neointimal formation and cell proliferation, we focused on three groups: control, CU10 and CI10.
Inhibitory Effect of Citrus Fruits on Cell Proliferation We observed that drinking citrus fruit juice decreased the neointima area, with a decrease in PCNA labeling index in both the intima and media (Fig. 2A and 2B). The increase in cell proliferation after cuff-placement was markedly attenuated in CI10, but not in CU10 compared with C.
Inflammatory Cytokine Levels Were Not Attenuated by Citrus Fruits We assessed the effect of drinking citrus fruit juice on mRNA levels of inflammatory cytokines in the femoral artery 7 days after cuff placement (Fig. 3). The increases in various inflammatory cytokines such as monocyte chemotactic protein-1, interleukin-6 (IL-6), IL-1β and tumor necrosis factor-α in response to vascular injury did not differ significantly among C, CU10 and CI10.
Inhibitory Effect of Citrus Fruits on Superoxide Anion Production Next, we examined the effect of drinking citrus fruit juice on superoxide anion production in the femoral artery 7 days after cuff placement (Fig. 4A and 4B). The increase in superoxide anion production determined by dihydroethidium staining was markedly attenuated in CI10, but not in CU10 compared with C.
Inhibitory Effect of Citrus Fruits on ERK Activation We investigated ERK activation in the injured artery 7 days after cuff placement (Fig. 5). The increase in expression of phosphorylated ERK determined by immunohistochemical staining and immunoblot using anti-phosphorylated ERK antibody. The increase in phosphorylated ERK positive cells in neointima determined by immunohistochemical staining was markedly attenuated both in CU10 and CI10 compared with C (Fig. 5A and 5B). Consistent with these immunohistochemical results, ERK activity determined with Western blotting was markedly attenuated in CI10 and CU10 compared with C (Fig. 5C). However, significant difference in the inhibitory effects between CU10 and CI10 on ERK activation was not observed.
PLOS ONE | DOI:10.1371/journal.pone.0117616 February 18, 2015
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Drinking Citrus Fruit Juice Inhibits Vascular Remodeling
Fig 1. Effect of drinking citrus fruit juice on neointima formation. Male C57BL6 mice were divided into five groups as follows: 1) Control (water) (C), 2) 10% Citrus unshiu (CU) juice (CU10), 3) 40% CU juice (CU40), 4) 10% Citrus iyo (CI) juice (CI10), and 5) 40% CI juice (CI40). After drinking them for 2 weeks from 8 weeks of age, cuff injury was induced by polyethylene cuff placement around the femoral artery. Samples were prepared from cuffed-femoral arteries of C57BL/6J mice as described in Methods. A, Representative photos of neointimal area in cross-sections of femoral artery with elastic van Gieson staining 14 days after cuff placement at 100x magnification. B, Higher magnified photos at 400x magnification described as squares in Figure A. Scale bars show 50 μm in each photo. C, Quantitative analysis of neointimal area in injured femoral artery. Values are mean ± SEM (n = 6 for Cuff (-), n = 8 for other groups). *p
